Pharmaceutical angiostatic dipeptide compositions and methods of use thereof

ABSTRACT

Disclosed are methods of inhibiting neovascularization in a subject by administering to the subject a pharmaceutical preparation of R&#39;-Glu-Trp-R&#39;&#39;.

This application is a continuation-in-part of copending Ser. No.08/538,701, filed Oct. 3, 1995, incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to pharmaceutical compositionscontaining peptides having angiostatic properties and more particularlyto pharmaceutical compositions of tryptophan-containing dipeptides andmethods of use thereof.

Neovascularization, the genesis of new blood vessels, is triggered earlyin embryogenesis and also during wound healing, tissue remodeling andprobably in the normal course of maintenance of the vascular system.Processes involved in neovascularization include at least endothelialcell and pericyte activation; basal lamina degradation; migration andproliferation of endothelial cells and pericytes; formation of a newcapillary vessel lumen; appearance of pericytes around the new vessels;development of a new basal lamina; capillary loop formation; persistenceof involution with differentiation of the new vessels; capillary networkformation; and, eventually, of the network organization into largermicrovessels.

Certain cytokines are known to down-regulate neovascularization,including interleukin-12 (IL-12), transforming growth factor-β (TGF-β),interferon-α (IFN-α) and platelet factor 4 (PF-4). However, clinicalexperience with cytokine therapy has proven problematic due to thetoxicity of certain of these compounds.

There are a number of pathologic conditions in which angiogenesis eitherplays a role in or is involved directly in different sequelae of thedisease. These include, for example, neovascularization of tumors incancer; creation of hemangeomas; neovascularization associated withvarious liver diseases; angiogenic dysfunction related to an excess ofhormone; neovascular sequelae of diabetes; neovascular sequelae tohypertension; neovascularization in post-recovery cerebrovascularaccident; neovascularization due to head trauma; neovascularization inchronic liver infection; restenosis following angioplasty; andneovascularization due to heat or cold trauma.

While angiogenesis is undoubtedly required for maintenance of a healthyvascular system, clinical medicine would appreciate the availability ofa non-toxic treatment for temporarily down-regulatingneovascularization, i.e., inducing a temporary angiostasis.

SUMMARY OF THE INVENTION

L-Glu-L-Trp has been known to stimulate the production of immune cellsand to normalize their numerical relationship in immune deficiencyconditions. (See, e.g., WO 89/06134, WO 92/17191 and WO 93/08815.)However, it has been discovered here that the dipeptide also hasangiostatic activity independent of its effect in immune deficiencyconditions. The results of studies in vitro showed that low levels ofL-Glu-L-Trp dipeptide inhibit neovascularization of chickenchorioallantoic membranes during embryogenesis. In animal studies,L-Glu-L-Trp inhibited neovascularization of Lewis lung tumor wheninjected intradermally in C57BL/6 mice, and inhibited growth of Sarcoma180 in Swiss-Webster mice.

Accordingly, this invention provides methods of treating a subjecthaving a pathologic condition involving neovascularization byadministering a pharmaceutical preparation comprising an R′-Glu-Trp-R″dipeptide and a pharmaceutically acceptable carrier to the subject in anamount effective to inhibit neovascularization.

In particular, this invention provides methods of treating subjectshaving the following pathologic conditions involving neovascularization:hemangiomas; vascularized malignant and benign tumors, including astumors of the meninges, intracerebral tumors, sarcomas, osteosarcomas,soft tissue tumors such as those of the esophagus and trachea;substance-induced neovascularization of the liver, including thatinduced secondary to ingestion of drug, alcohol or substances of abuse;angiogenic dysfunction related to an excess of hormone, e.g., estrogen;neovascular sequelae of diabetes, such as central serouschorioretinopathy; neovascular sequelae to hypertension;neovascularization in post-recovery cerebrovascular accident;neovascularization due to head trauma; chronic liver infection (e.g.,chronic hepatitis); restenosis following angioplasty; andneovascularization due to heat or cold trauma, such as burn orfrostbite. The dipeptide exhibits this activity both in subjects withhealthy immune systems, i.e., who are not immune compromised, as well asthose subjects who are immune compromised.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutical Glu-Trp preparationscomprising an R′-Glu-Trp-R″ dipeptide and a pharmaceutically acceptablecarrier. R′-Glu-Trp-R″ dipeptide, as used herein, refers to thedipeptide L-Glu-L-Trp and derivatives or analogues thereof.

As used herein, a derivative of the R′-Glu-Trp-R″ dipeptide includesthose in which the dipeptide is derivatized by the covalent attachmentof a moiety at R′ and/or R″. This includes, for example,pharmaceutically acceptable salts of the dipeptide, amides, imides,esters, anhydrides, ethers, methyl or ethyl-alkyl esters, alkyl, aryl ormixed alkyl/aryl derivatives wherein the formula weight is less thanabout 5000 Daltons or less than 1000 Daltons, multimeric or cyclicversions of the dipeptide and peptides of fewer than about 20 aminoacids or less than about 10 amino acids that include glu-trp withintheir amino acid sequence. Representative examples include HEW, EWEW,GEW, EWKHG, EWKKHG, EW-NH-NH-GHK-NH₂, Ac-L-Glu-L-Trp-OH, Suc-EW, Cpr-EW,But-EW, RKEWY, RKEW, KEWY, KEW, pEW.

As used herein, analogs of R′-Glu-Trp-R″ dipeptide include those inwhich L-amino acids are substituted for D-amino acids, such asL-Glu-D-Trp, D-Glu-L-Trp or D-Glu-D-Trp, and analogs of tryptophan suchas 5-hydroxy-tryptamine, 5-hydroxy-indol-acetic acid and pyrole analogsin which the nitrogen in the pyrole ring is replaced with carbon.

L-Glu-Trp-L presently is the most preferred R′-Glu-Trp-R″ dipeptide.L-Glu-L-Trp is also referred to herein interchangeably as “EW” and “EWdipeptide”, using the single letter convention wherein the first namedamino acid is the amino terminus and the last named amino acid is thecarboxyl terminus.

As used herein, “neovascularization” refers to the generation of newblood vessels. The process by which new blood vessels are formed mayinvolve processes of endothelial cell and pericyte activation; basallamina degradation; migration and proliferation (i.e., cell division) ofendothelial cells and pericytes; formation of a new capillary vessellumen; appearance of pericytes around the new vessels; development of anew basal lamina; capillary loop formation; persistence of involution,and differentiation of the new vessels; and, capillary network formationand, eventually, organization into larger microvessels. As referred toherein the process of endothelial cell proliferation, e.g., in avascular bud, is termed “angiogenesis” and is related toneovascularization as a subprocess. Representative clinical diagnosticmanifestations of diseases have been classified and codified(International Classification of Diseases, ICD-9-CM, Washington, D.C.1989.) Representative laboratory indicia of neovascularization include(but are not limited to) data e.g. collected in angiograms, CAT scansand sonograms, as well as visual examination by endoscopic and/orcapillaroscopic procedures.

As used herein the terms “angiostasis,” “angiostatic” and “inhibition ofneovascularization” mean that the rate or extent of neovascularizationin a tissue is decreased from a pre-treatment value to a post-treatmentvalue. Angiostasis may be determined using laboratory or clinicalindicia of disease activity, above. Angiostasis may involve inhibitingone or more subprocesses involved in neovascularization, e.g.,endothelial or vascular smooth muscle cell proliferation or migration.

As used herein, a “pathologic condition involving neovascularization”refers to a pathologic condition in which neovascularization or the riskof it is a component. This includes, without limitation, pathologies inwhich neovascularization is the primary pathology, such as hemangiomas;pathologies in which neovascularization is not the primary pathology butcontributes to it, such as neovascularization of tumors; and pathologiesin which neovascularization is a sequela of the primary disease, such ascentral serous retinopathy in diabetes.

As used herein, the term “subject” refers to a mammal, including humanand non-human primates, domestic animals and livestock, fur bearinganimals, and the like, e.g., dogs, cats, rodents, birds, horses, cows,pigs, fish, and the like. Embodiments of the invention encompasstherapeutic and prophylactic treatment methods for use in subjects inneed thereof.

As used herein the term “immune compromised” refers to a person having alower than normal number of one or more immune cells, such as NK cells,T4 or T8 T-lymphocytes, B-lymphocytes, or phagocytes, as measured bystandard clinical diagnostic indicia. It also includes individualshaving diminished function of immune cells as determined by standardfunctionality testing of such cells, e.g., production ofimmunoglubulins, chemotaxis, mixed leukocyte reaction or delayedhypersensitivity assay. Immune compromised individuals often presentwith unusual or unexpected opportunistic infections.

“Polypeptide” is intended to mean a serial array of amino acids of morethan 16 and up to many hundreds of amino acids in length, e.g., aprotein.

“Abnormal” as used herein refers to laboratory indicia ofneovascularization that are outside of the range of values recorded inhealthy individuals.

“Normalized” as used herein refers to changes in laboratory or clinicalindicia of neovascularization that are, following treatment, returned towithin the normal range of values recorded for normal healthy subjects.A subject without a vascular defect and without a known deficiency inany coagulation, fibrinolytic, or vascular system is referred to hereininterchangeably as “a normal subject”.

As used herein, the terms “modulator” and “modulating” mean the agentand process of decreasing neovascularization or angiogenesis in a normalsubject or in a compromised subject.

R′-Glu-Trp-R″ treatment is intended to mean a method of delivering to asubject in need thereof a pharmaceutical preparation of R′-Glu-Trp-R″with the aim of inducing a decrease in the rate or extent ofneovascularization or angiogenesis.

In one presently preferred embodiment, an R′-Glu-Trp-R″ pharmaceuticalpreparation is administered to a cancer patient in an amount and for atime sufficient to decrease one or more clinical or laboratory indiciaof neovascularization or angiogenesis, thereby effecting improvement inthe clinical condition of the patient so treated. This method decreasesneovascularization in the tumor, inhibiting blood supply to the tumorand, thereby, inhibiting growth of the tumor.

In a preferred embodiment a treatment regimen consists of administeringa dose of about 0.5 μg per 1 kilogram body weight to about 1 mg per 1 kgbody weight daily over a period of 1 day to about 30 days to thesubject. In preferred embodiments the subject dose is administeredeither as a single daily intramuscular dose of the R′-Glu-Trp-R″pharmaceutical preparation (intramuscularly), or as a single dailyintranasal dose of the R′-Glu-Trp-R″ pharmaceutical preparation(intanasally). The subject dose is preferably formulated as a sterile,injectable, inhalant, nose drop, or mucosal spray solution containingabout 0.001% to about 0.01% of the R′-Glu-Trp-R″ pharmaceuticalpreparation. Alternatively, the formulation of the R′-Glu-Trp-R″pharmaceutical preparation may preferably be incorporated into a unitdose delivery form e.g., a tablet, a suppository, a capsule, an eyefilm, or into a paste or ointment, e.g., a toothpaste, a dermalointment, or water-soluble cream base. A most preferred unit dose formis for delivery of about 0.01 mg of the R′-Glu-Trp-R″ pharmaceuticalpreparation.

The subject methods of the invention find a variety of prophylactic andtherapeutic uses in treatment of pathophysiologic conditions in humansand domestic animals. In certain embodiments the methods of theinvention find use during in vitro maintenance of endothelial cellcultures and vascular tissues such as may occur prior to autologous orallogenic grafting. The methods involve maintaining endothelial cellcultures or vascular tissue cultures in vitro by culturing the tissuesin a culture medium comprising an R′-Glu-Trp-R″ compound. The subjectmaintenance method has the advantage of maintaining vascular tissues andreducing inflammatory alterations triggered by the tissue traumaoccurring during surgical removal and storage in tissue culture.

In a representative prophylactic treatment regimen, the subjectcompositions of the invention are administered to a patient susceptibleto or otherwise at risk for developing neovascularization, e.g., inpost-surgical use to prevent neovascularization of a recurring primarytumor or it metastatic cells. “Prophylactically effective dose” is usedherein to mean an amount sufficient to produce an angiostatic effect ata tissue site, wherein the amount will depend on the patient's state ofhealth and weight, but will generally fall within the ranges describedherein for therapeutic use. Prophylactic administration may beparticularly desirable for subjects that are at risk of disease sequelaeinvolving neovascularization or angiogenesis as a complication, e.g.,diabetic retinopathy.

Embodiments of the invention include therapeutic treatment regimenswherein an R′-Glu-Trp-R″ pharmaceutical preparation is administeredalone, or in combination with a second pharmaceutical agent, i.e.,“combined therapy”. Representative combined therapies include those inwhich an R′-Glu-Trp-R″ composition is administered with one or moreantibiotics, anti-inflammatory agent, or chemotherapeutic compounds. Thesubject compositions may be administered either in conjunction with thesecond treatment modalities, or separately, e.g. at different times orin different syringes or tablets. Often, R′-Glu-Trp-R″ is administeredin a combined therapy with anti-inflammatory agents, antihistamines,chemotherapeutic agents and the like. Illustrative combined treatmentswith R′-Glu-Trp-R″ may include, e.g., anti-inflammatory agents wellknown in the art.

Illustrative combined treatments with R′-Glu-Trp-R″ may also includeadministration of a vasoactive drug as the second agent. Representativevasoactive drugs so active include drugs in the class of angiotensinconverting enzyme (ACE) inhibitors, potassium channel openers (PCO) andthe like.

Illustrative combined cancer treatments with R′-Glu-Trp-R″ includeadministration of a chemotherapeutic agent as the second agent.Treatments with R′-Glu-Trp-R″ may be effective to decrease undesirableside-effects associated with a corticosteroid therapy, e.g.,neovascularization. Representative chemotherapeutic agents are wellknown in the art.

Skilled practitioners will adjust the timing and dosage to fit theclinical symptoms of the patients. Such knowledge has been accumulatedover decades, and is reported in the medical literature as well asmedical texts. The timing of when to start the subject methods incombination or single agent therapy rests on the physician's clinicaljudgment.

Empirical therapy is a therapy designed to treat the most common orlikely causative agent based on historic, demographic, and epidemiologicinformation. Empirical therapy may often include use of multipletherapeutic agents designed to cover a wide range of therapeuticpossibilities. When laboratory test data are available the choice oftherapy may be adjusted to more particularly treat the disease. Becausetreatment of clinical syndromes is very often initiated empirically.Rather, a new therapeutic method must be tested for a particularclinical syndrome.

In the art of pharmaceutical drug development, preclinical studies of atherapy evaluate the therapy's effects on not just one condition, but onmultiple agents or conditions of interest. The results of the various(sometimes equivocal) studies are weighed as to the benefits and risksof the particular therapy given the medical knowledge of the risksassociated with a particular disease.

It is common that not all patients with a syndrome are cured by a singletherapy, but instead, that a subset of patients may exist wherein thetherapy has a positive and favorable result. Examples of clinicalsyndromes in which subsets of patients may find favorable outcomes fromthe subject therapies of the invention are disclosed in the followingseveral paragraphs.

The pharmaceutical compositions of the invention are intended forparenteral, topical, subcutaneous, intramuscular, intrathecal, oral,intranasal, intraperitoneal or local administration (e.g. on the skin ina cream), or prophylactic and/or therapeutic treatment. Preferably, thecompositions of the present invention are administered parenterally,intramuscularly or intranasally. The subject R′-Glu-Trp-R″ compositionsherein have the advantage of providing the desired effects at very lowdosage levels and without toxicity. Thus, a purpose of therapy in anacute setting may be to rapidly increase the concentration ofR′-Glu-Trp-R″ in a tissue, e.g., by bolus intravenous injection orinfusion. Alternatively, in other cases it may desirable to deliverR′-Glu-Trp-R″ over a longer period of time.

The subject compositions containing R′-Glu-Trp-R″ may be formulated in amanner that allows absorption into the blood stream. The presentcompositions are vascular modulators that induce changes at the cellularlevel that subsequently effect changes in cellular processes that nolonger are dependent on the presence of the composition. It has beenobserved that the effects of the peptide may be long lasting, i.e., forweeks to months, despite the rather rapid degradation of the peptide,e.g. within 5 minutes. Although the subject R′-Glu-Trp-R″ compounds arethemselves water-soluble at the low concentrations in which they areusually employed, they are preferably used in the form of their acid oralkaline salts formed with pharmaceutically acceptable agents, e.g.,acetic, citric, maleic, succinic acid, sodium, potassium, ammonium, orzinc. Freely-soluble salts of the subject R′-Glu-Trp-R″ compositions mayalso be converted to salts of low solubility in body fluids e.g., bymodification with a slightly water-soluble pharmaceutically acceptablesalt like tannic or palmoic acid, or by inclusion in a time-releaseformulation with covalently coupling to a larger carrier, or inclusionin timed-release capsule and the like.

The subject R′-Glu-Trp-R″ pharmaceutical preparations may be used asfree peptides or in the form of a water soluble pharmaceuticallyacceptable salts, such as a sodium, potassium, ammonium or zinc salt. Itwill be understood that the subject dipeptides may be administered withother active ingredients which independently impart an activity to thecomposition. Pharmaceutically acceptable salts may be convenientlyprepared from an R′-Glu-Trp-R″ dipeptide (or its agonist) byconventional methods. Thus, such salts may be, for example, prepared bytreating R′-Glu-Trp-R″ dipeptide with an aqueous solution of the desiredpharmaceutically acceptable metallic hydroxide or other metallic baseand evaporating the resulting solution to dryness, preferably underreduced pressure in a nitrogen atmosphere. Alternatively, a solution ofR′-Glu-Trp-R″ dipeptide may be mixed with an alkoxide to the desiredmetal, and the solution subsequently evaporated to dryness. Thepharmaceutically acceptable hydroxides, bases, and alkoxides includethose with cations for this purpose, including (but not limited to),potassium, sodium, ammonium, calcium, and magnesium. Otherrepresentative pharmaceutically acceptable salts include hydrochloride,hydrobromide, sulfate, bisulfate, acetate, oxalate, valarate, oleate,laurate, borate, benzoate, lactate, phosphate, tosulate, citrate,maleate, fumarate, succinate, tartrate, and the like.

For parenteral administration the present invention providespharmaceutical preparations for parenteral administration which comprisea solution of R′-Glu-Trp-R″ dipeptide, or polymeric, multimeric, orcyclic forms or derivative thereof, dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers may be used, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like, including proteins and/or glycoproteins forenhanced stability, such as albumin, lipoprotein, globulin, and thelike. These compositions may be sterilized by conventional, well knownsterilization techniques. The resulting aqueous solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile aqueoussolution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc. It may be desirable to stabilize R′-Glu-Trp-R″dipeptides, analogs, derivatives, agonists, and the like to increasetheir shelf life and pharmacokinetic half-life. Shelf life stability isimproved by adding excipients such as: a) hydrophobic agents (e.g.,glycerol); b) sugars (e.g., sucrose, mannose, sorbitol, rhamnose,xylose); c) complex carbohydrates (e.g., lactose); and/or d)bacteriostatic agents. Pharmacokinetic half-life of peptides is modifiedby coupling to carrier peptides, polypeptides, and carbohydrates bychemical derivatization (e.g., by coupling side chain or N- orC-terminal residues), or chemically altering the amino acid to anotheramino acid (as above). Pharmacokinetic half-life and pharmacodynamicsmay also be modified by: a) encapsulation (e.g., in liposomes); b)controlling the degree of hydration (e.g., by controlling the extent andtype of glycosylation of the peptide); and, c) controlling theelectrostatic charge and hydrophobicity of the peptide.

The R′-Glu-Trp-R″ dipeptide containing compositions according to thepresent invention may be administered in a compatible pharmaceuticalsuitable for parenteral administration (e.g., intravenous, subcutaneous,intramuscular). The preparations may be subjected to conventionalpharmaceutical operations, such as sterilization, and may containadjuvants, such as preservatives, stabilizers, wetting agents and thelike.

The R′-Glu-Trp-R″ dipeptide compositions are typically biologicallyactive at a dose of about 0.5 μg/kg to about 1 mg/kg, preferably about 1μg/kg to about 50 μg/kg. The concentration of the R′-Glu-Trp-R″dipeptides in pharmaceutical compositions can vary, i.e., from about0.001% to as much as 15 or 20% by weight and will be selected primarilyby fluid volumes, viscosities, etc., in accordance with the particularneeds of the treatment and mode of administration to a patient. Whenutilized intramuscularly as an injection solution with the activeingredient in a amount effective to inhibit neovascularization, e.g.,about 0.001 to 0.01% by weight of R′-Glu-Trp-R″. If prepared in the formof a tablet, capsule or suppository, it is preferred that the activeingredient be present in an amount of about 0.1 mg of R′-Glu-Trp-R″ pertablet, suppository or capsule. The pharmaceutically acceptable vehiclefor this injection form may be any pharmaceutically acceptable solventsuch as 0.9% aqueous sodium chloride, distilled water, Novocainesolution, Ringer's solution, glucose solution, and the like. In suchform, the capsule, suppository or tablet may also contain otherconventional excipients and vehicles such as fillers, starch, glucose,etc. In topical preparations, the R′-Glu-Trp-R″ dipeptides are generallycontained in urea-based emollients, petroleum-based ointments, and thelike at concentrations of about 0.1 to 10,000 parts per million,preferably about 1 to 1000 parts per million, and most preferably about10 to 100 parts per million. Actual methods for preparing parenterally,orally, and topically administrable compounds will be known or apparentto those skilled in the art and are described in detail in, for example,Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), which is incorporated herein by reference.

Intramuscular and intranasal routes are preferred for administration ofthe subject R′-Glu-Trp-R″ compositions. One preferred dosage of thesubject composition for intramuscular administration is about 50 μg to100 μg R′-Glu-Trp-R″ per dose for adults (for a 300 μg to 1000 μg totaltreatment therapy); for infants up to 1 year old about 10 μg per dose,for infants 1 to 3 years old about 10 μg to 20 μg per dose; for infants4 to 6 years old about 20 μg to 30 μg per dose, for children 7 to 14years old about 50 μg per dose. All of the foregoing dosages are usefulfor a treatment of 3 to 10 days, depending upon the needs of thepatient. The treatment may be repeated as needed, usually within 1 to 6months. In another preferred embodiment a treatment dose of about 10μg/kg to about 1 mg/kg of a R′-Glu-Trp-R″ pharmaceutical preparationadministered to a subject daily over a period of about 6 days to about10 days but optionally at the discretion of the attending physician forup to about 30 days. In one preferred course of therapy R′-Glu-Trp-R″ isadministered im daily at a dosage of 1-100 μg/kg for 5-7 days, followedby a 1-6 month intermission before repeating the same injection regimen.

The R′-Glu-Trp-R″ composition may be administered alone or formulatedwith pharmaceutically acceptable carriers, in either single or multipledoses. Suitable pharmaceutical carriers include inert solid diluents orfillers, sterile aqueous solutions, and various nontoxic organicsolvents. The pharmaceutical compositions formed by combiningR′-Glu-Trp-R″ dipeptide with a pharmaceutically acceptable carrier andan optional antibiotic. The subject combination therapeutic agents arethen readily administered in a variety of dosage forms such as tablets,lozenges, syrups, injectable solutions, and the like. Combinationtherapeutic agents may also include R′-Glu-Trp-R″ dipeptide, e.g.,L-Glu-L-Trp, in the same unit dosage form. Pharmaceutical carriers can,if desired, contain additional ingredients such as flavorings, binders,excipients, and the like.

Thus, for purposes of oral administration, tablets containing variousexcipients such as sodium citrate, calcium carbonate, and calciumphosphate may be employed along with various disintegrants such asstarch, and preferably potato or tapioca starch, alginic acid, andcertain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin, and acacia. Additionally,lubricating agents, such as magnesium stearate, sodium lauryl sulfate,and talc are often useful for tableting purposes. Solid compositions ofa similar type may also be employed as fillers in salt and hard-filledgelatin capsules. Preferred materials for this purpose include lactoseor milk sugar and high molecular weight polyethylene glycols. Whenaqueous suspensions of elixirs are desired for oral administration, theessential active R′-Glu-Trp-R″ dipeptide ingredients therein may becombined with various sweetening or flavoring agents, colored matter ordyes, and if desired, emulsifying or suspending agents, together withdiluents such as water, ethanol, propylene glycol, glycerin, andcombinations thereof.

For parenteral administration, solutions of R′-Glu-Trp-R″ in sesame orpeanut oil or in aqueous polypropylene glycol may be employed, as wellas sterile aqueous saline solutions of the corresponding water solublepharmaceutically acceptable metal salts previously described. Such anaqueous solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal injection. The sterileaqueous media employed are all readily obtainable by standard techniqueswell known to those skilled in the art. Additionally, it is possible toadminister the aforesaid compounds topically (e.g., through a placedcatheter) using an appropriate solution suitable for the purpose athand.

An amount adequate to effect a therapeutic result in more than 50% ofsubjects so treated is defined as a “therapeutically effective dose.”Treatment of acute conditions generally will occur over about 3-10 days.Treatment of chronic conditions or prophylactic treatments have the samecourse, but can be repeated after as long as about 1-6 months or longer.In some instances, it may be desirable to administer the compositionsintermittently on a daily basis for periods of about 2 to about 20 days,preferably about 3 to about 14 days, more preferably about 4 to about 10days which are repeated at least about 15 days, preferably about 20 daysor as much as about 1 to 6 months or more.

The route of delivery of a R′-Glu-Trp-R″ composition is determined bythe disease and the site where treatment is required. For topicalapplication it may be desirable to apply the R′-Glu-Trp-R″ compositionat the local site (e.g., by placing a needle into the tissue at thatsite) or by placing an impregnated bandage e.g. at a tumor sitefollowing surgical removal; while for other diseases it may be desirableto administer the R′-Glu-Trp-R″ compositions systemically. For otherindications the R′-Glu-Trp-R″ compositions and the like may be deliveredby intravenous, intraperitoneal, intramuscular, subcutaneous,intranasal, and intradermal injection, as well as, by intrabronchialinstillation (e.g., with a nebulizer), transdermal delivery (e.g., witha lipid-soluble carrier in a skin patch), or gastrointestinal delivery(e.g., with a capsule or tablet).

In general, the acid addition salts of the subject R′-Glu-Trp-R″composition, e.g., L-Glu-L-Lys, compositions with pharmaceuticallyacceptable acids will be biologically equivalent to the subjectR′-Glu-Trp-R″ compositions themselves.

The preferred therapeutic compositions, inocula, routes, and dosage willof course vary with the clinical indication. For intramuscular injectionthe inocula is typically prepared from a dried peptide (or peptideconjugate) by suspending the peptide in a physiologically acceptablediluent such as water, saline, or phosphate-buffered saline. Somevariation in dosage will necessarily occur depending upon the conditionof the patient being treated, and the physician will, in any event,determine the appropriate dose for the individual patient. The effectiveamount of peptide per unit dose depends, among other things, on the bodyweight, physiology, and chosen inoculation regimen. A unit does ofpeptide refers to the weight of peptide without the weight of carrier(when carrier is used). An effective treatment will be achieved when theconcentration of R′-Glu-Trp-R″ dipeptide, e.g., L-Glu-L-Trp, at a tissuesite in the micro-environment of the cells approaches a concentration of10⁻⁵ M to 10⁻⁹ M. Skilled practitioners can make use of clinical andlaboratory indicia (above) to monitor patient response to the subjecttherapy and adjust the dosage accordingly. Since the pharmacokineticsand pharmacodynamics of R′-Glu-Trp-R″ dipeptides, agonists, antagonists,and the like will vary in different patients, a most preferred methodfor achieving a therapeutic concentration in a tissue is to graduallyescalate the dosage and monitor the clinical and laboratory indicia(above). The initial dose, for such an escalating dosage regimen oftherapy, will depend upon the route of administration. For intravenousadministration, of R′-Glu-Trp-R″ dipeptide with an approximate molecularweight of 200 to 400 daltons, an initial dosage of approximately 0.5μg/kg body weight is administered and the dosage is escalated at 10-foldincreases in concentration for each interval of the escalating dosageregimen.

If presented in the form of a tablet, capsule or suppository it ispreferred that the active ingredient be present in an amount of about0.1 mg per tablet, suppository or capsule. If presented in such form,the capsule, suppository or tablet may also contain other conventionalexcipients and vehicles such as fillers, starch, glucose, etc.

Conveniently, the subject R′-Glu-Trp-R″ dipeptide is synthesized by anyof a number of automated techniques that are now commonly available.Generally speaking, these techniques involve stepwise synthesis bysuccessive additions of amino acids to produce progressively largermolecules. The amino acids are linked together by condensation betweenthe carboxyl group of one amino acid and the amino group of anotheramino acid to form a peptide bond. To control these reactions, it isnecessary to block the amino group of one amino acid and the carboxylgroup of the other. The blocking groups should be selected for easyremoval without adversely affecting the peptides, i.e., by racemizationor by hydrolysis of the formed peptide bonds. Amino acids withcarboxyl-groups (e.g., Asp, Glu) or hydroxyl-groups (e.g., Ser,homoserine, and tyrosine) also require blocking prior to condensation.

A Wide variety of procedures exist for synthesis of peptides,solid-phase synthesis usually being preferred. In this procedure anamino acid is bound to a resin particle, and the peptide generated in astepwise manner by successive additions of protected amino acids to thegrowing chain. Modifications of the technique described by Merrifieldare commonly used. In an exemplary automated solid-phase method,peptides are synthesized by loading the carboxy-terminal amino acid ontoan organic linker (e.g., PAM, 4-oxymethyl phenylacetamidomethyl)covalently attached to an insoluble polystyrene resin that iscross-linked with divinyl benzene. Blocking with t-Boc is used toprotect the terminal amine, and hydroxyl- and carboxyl-groups arecommonly blocked with O-benzyl groups. Synthesis is accomplished in anautomated peptide synthesizer (Applied Biosystems, Foster City, Calif.,e.g., Model 430-A). Following synthesis the product may be removed fromthe resin and blocking groups removed using hydrofluoric acid ortrifluoromethyl sulfonic acid according to established methods (Bergot,B. J. and S. N. McCurdy, Applied Biosystems Bulletin, 1987). A routinesynthesis can produce 0.5 mmole of peptide-resin. Yield followingcleavage and purification is approximately 60 to 70%. For example, anamino and side chain protected derivative of an activated ester of Glxis reacted with side-group protected Trp, attached to the solid phase atits C-terminus. After elimination of the alpha-amino protecting group,the peptide maybe cleaved from the solid phase or another amino acidadded in a similar fashion. Additional amino acids are serially added.The peptides are cleaved by highly acidic cleavage that also typicallyremoves protecting groups.

The peptides may then be isolated and lyophilized and stored for futureuse. Suitable techniques of peptide synthesis are described in detail inStewart and Young, Solid Phase Peptide Synthesis, 2d edition, PierceChemical Company, 1984; and Tam et al., J. Am. Chem. Soc., 105:6442(1983), both of which are incorporated herein by reference.

Purification of the product peptides is accomplished for example bycrystallizing the peptide from an organic solvent such as methyl-butylether, followed by dissolving in distilled water, and dialysis (ifgreater than about 500 molecular weight), thin layer chromatography, gelchromatography, lyophilization, or reverse HPLC (e.g., using a C18column with 0.1% trifluoroacetic acid and acetonitrile as solvents) ifless than 500 molecular weight. Purified peptide is lyophilized isstored in a dry state until use. A representative R′-Glu-Trp-R″pharmaceutical preparation is the purified dipeptide L-Glu-L-Trp, whichcomprises a white powder (if lyophilized; otherwise, it is crystalline),soluble in water, DMF; insoluble in chloroform and ether.[alpha22_(D)=+12.6; C=0.5 H₂O. R_(f)=0.65 (butanol: acetic acid:water=3:1:1). UV (275±5 nm, max). NMR (500 MHz): 0.001 mol/l of thepeptide solution, Trp (3.17; 3.37; 4.57; 7.16; 7.24; 7.71; 7.49); Glu(1.90; 1.96; 2.21; 3.72).

Typically an amino and side chain protected derivative of an activatedester of glutamic acid is reacted with protected L-tryptophan. Afterelimination of the protecting groups and conventional purification, suchas by thin layer or GL chromatography, the peptide may be purified suchas by, lyophilization, gel purification, and the like.

While not wishing to be tied to any particular mechanism of action, itis believed possible that the subject tryptophan-containing peptides mayreversibly associate with specific cellular EW receptors on endothelialcells, one such receptor being defined as the ubiquitous “CD2” cellsurface determinant present also on lymphocytes, endothelial cells andcertain epithelial cells. It is thought possible that binding of EWdipeptide to CD2 (and other EW receptors) triggers a conformationalchange in the receptor that may initiate up-regulation of adenylatecyclase and increased intracellular cAMP. It is presently believepossible that L-Glu-L-Trp exerts its effects by down-regulating cellularmechanisms by which inflammatory mediators such as TNF-α and IL-1trigger endothelial cell and pericyte activation and proliferation.Activation results in changes in cell surface expression of adhesinsinvolved in binding inflammatory cells in vasculitis, whileproliferation is involved in neovascularization. L-Glu-L-Trpdown-regulation of inflammatory mediator-induced endothelial effects mayinvolve dephosphorylation of one or more cellular tyrosine kinases. Itis considered likely that such down-regulation may result in changes insynthesis or cell surface expression of endothelial adhesins, selecting,and/or integrins, e.g., ELAM, VCAM, and the like. The latter cellularchanges induced by tryptophane-containing dipeptides may result in adecreased ability of inflammatory cells (e.g., lymphocytes, neutrophils,and/or monocytes) to localize at sites of vasculitis.

As used herein the symbols for amino acids are according to theIUPAC-IUB recommendations published in Arch. Biochem. Biophys. 115:1-12, 1966 with the following single letter symbols for the amino acids:L, Leu, Leucine; V, Val, Valine; Y, Tyr, Tyrosine; D, Asp, AsparticAcid; W, Trp, Tryptophan; P, Pro, Proline; I, Ileu, Isoleucine; G, Gly,Glycine; M, Met, Methionine; E, Glu, Glutamic Acid; T, Thr, Threonine;K, Lys, Lysine; N, Asn, Asparagine; R, Arg, Arginine; Q, Gln, Glutamine;A, Ala, Alanine; C, Cys, Cysteine; S, Ser, Serine; F, Phe,Phenylalanine; H, His, Histidine; C, Cys, Cysteine; S, Ser, Serine.

The following examples are provided to further elucidate the invention,but are not intended to restrict the invention in scope or spirit in anyway.

EXAMPLE 1 Lack of Mutagenicity and Toxicity of L-Glu-L-Trp:Pharmacokinetics and Biodistribution

*Note that general materials and methods used in Examples, below, appearat the end of the Examples section and immediately before the citations.

Protocol A Acute Toxicity Studies

Summary: L-Glu-L-Trp when injected im at dosages calculated to be about10,000-times a therapeutic dosage were non toxic in mice, guinea pigs,chickens, and dogs as determined by monitoring general condition,behavior, movements, cardiac and respiratory physiology, and grosspathology.

Protocol B Chronic Toxicity Studies

Summary: L-Glu-L-Trp when injected daily as a single im or iv for aperiod of 28 days was without adverse effects as determined bymonitoring behavior, feeding, body weight, coat condition, mucousmembranes, red and white cell blood counts, cardiac and respiratoryphysiology, liver and kidney function, and gross pathology. Kidneyfunction was determined by evaluation of diuresis after water-loading;for certain other experiments dogs and rats were sacrificed and examinedafter 10, 20, 30, and 60 days.

Protocol C Pharmacokinetics and Biodistribution: GLP Study

¹⁴C-radiolabeled L-Glu-L-Trp (110 μg/kg) was administered intranasallyto Sprague-Dawley rats. Blood and tissue samples were collected atdifferent 0.5, 2, 8 or 24 hours and the amount of intact L-Glu-L-Trp wasdetermined by HPLC. Tissue samples included packed red blood cells,white blood cells, liver, kidney, heart, lung spleen, thymus, brain,muscle, skin, fat, eye, ovaries, testes, submandibular lymph nodes, andthe gastrointestinal tract with contents. Intranasally administered¹⁴C-L-Glu-L-Trp was rapidly absorbed with a plasma C_(max) of 0.349μg*eq.*hr/g of the ¹⁴C. No intact compound was detected in blood at 30minutes to a sensitivity in the range of 5-101 ng/mL, suggesting a bloodhalf life of less than 30 minutes. Tissue elimination half-life wasdetermined to be 18.7 hr.

EXAMPLE 2 Inhibitory Effects of L-Glu-L-Trp on Angiogenesis inChorioallantoic Membrane (CAM) Assays

Briefly, eight-day chicken embryos were removed from eggs and placedinto sterile petri dishes. Individual filter paper disks were saturatedwith 7.5 μl of different stock solutions of L-Glu-L-Trp dissolved insterile 0.14M NaCl to achieve final test concentrations of 0.001, 0.01,0.1, 1.0, 10, 100, 500, and 1000 μg per disk. Disks were air dried andthen inverted onto the surface of the respective embryos. Embryovascularization was assessed after 48 hrs. of incubation using thegrading scale summarized in TABLE 1, below. TABLE 1 Scoring of ChickenEmbryo Vascularity: CAM Assay Percentage Inhibition Grade DescriptionInhibition 0 Not visibly difference than negative 0 control 1 Slightinhibition of vessel formation 25 2 Moderate inhibition of vesselformation 50 3 Near-complete inhibition of vessel 75 formation 4Complete inhibition of vessel formation 100

In this experiment saline served as a negative control and 10 μg/disk ofheparin served as a positive control. The pentapeptideTyr-Ala-Glu-Glu-Lys (TAEEK) served as a specificity control, (i.e., forpossible nonspecific effects of peptides on neovascularization at theconcentrations tested). Nine-12 test disks and a corresponding number ofdifferent embryos were employed for each test concentration along with82 (each) positive and negative control embryos. The results aresummarized in the following TABLE. TABLE 2 Results of Chicken Embryo CAMAssay Inhibition of Neovascularization Grade/ Test No. ConcentrationRange Grade/ Article Embryo (μg/disk) Inhibition Mean ± SD* Mean %Saline 82 0 0-0 0 ± 0 0 ± 0 Heparin 82 10 1-4 3.26 ± 0.73 81 ± 18L-Glu-L- 10 1000 2-4  3.3 ± 0.82 83 ± 20 Trp 10 500 1-4  2.4 ± 0.84 60 ±20 9 100 3-4 3.44 ± 0.73 85 ± 18 11 10 1-4 3.09 ± 1.14 78 ± 28 12 1 1-42.33 ± 0.89 58 ± 23 10 0.1 0-3  1.9 ± 0.88 48 ± 23 10 0.01 1-2  1.5 ±0.53 38 ± 13 10 0.001 0-2  1.3 ± 0.82 33 ± 20 TAEEK 10 1000 0-2  0.7 ±0.82 18 ± 20 10 500 0-1  0.3 ± 0.48  9 ± 13 9 100 0-2 0.67 ± 0.87 18 ±23 11 10 0-1 0.18 ± 0.40  5 ± 10 12 1 0-1 0.33 ± 0.49  8 ± 10 10 0.1 0-00 ± 0 0 10 0.01 0-0 0 ± 0 0 10 0.001 0-0 0 ± 0 0*Mean ± SD = mean ± standard deviation, n = 10 for test and n = 80 forsaline and heparin.

The results show a 30-88% decrease in vascularity in embryos treatedwith 10 ng-1000 μg of L-Glu-L-Trp in saline. The level of inhibitionachieved at the 10-1000 μg doses approximated that with heparin (10 μg).Although some presumed nonspecific effects of control pentapeptide TAEEKon embryo vascularity was observed at the higher doses (i.e., 100-1000μg), the effect was not as pronounced as that achieved with L-Glu-L-Trpand the presumptive nonspecific effect was not observed at lower doses.Taken together these results suggest an effect of L-Glu-L-Trp on theprocess of vessel formation in embryonic chicken tissues.

EXAMPLE 3 Inhibition of Neovascularization of Lewis Carcinoma

Lewis lung carcinoma cells (5×10⁷) when injected (0.1 ml) intradermallyinto both flanks of C57BL/6 mice (day 0) produce a visible highlyvascularized tumor nodules within 7 days. By excising the tumor thedegree of tumor vascularity may be determined microscopically bycounting the number of large vessels radiating from the tumor mass. Anindependent study was performed (as follows) at a GLP approved contractresearch organization.

Saline was used as a negative control and Cytoxan as a positive control.The positive control, Cytoxan (200 mg/kg), was administered only on day2. Test treatments with L-Glu-L-Trp were administered im on a dailybasis starting on day 1 after tumor injection and continuing for 5 days(i.e., through day 6). L-Glu-L-Trp was administered at doses of 125,250, 500, 1000, and 2000 μg/kg/dose. The negative control, saline, wasadministered ip on the same daily 5 day schedule. Ten mice (20 tumors)were evaluated at each dose of test or control agent. The results aresummarized in the following TABLE. TABLE 3 Inhibition of Lewis LungTumor Neovascularization No. Vessels Group No. Treatment Dose(μg/kg/dose) (Mean ± S.D.)* 1 None 0 19 ± 6  2 Cytoxan 200 9 ± 5* 3L-Glu-L-Trp 2000 17 ± 7  4 1000 12 ± 5*  5 500 9 ± 4* 6 250 7 ± 2* 7 1256 ± 3**Student-Newman-Keuls multiple pairwise comparison; statisticallydifferent than group 1 at the p < 0.05 level.

The results show a clear statistically significant inhibition ofneovascularization as a result of treatments with either Cytoxan orL-Glu-L-Trp. Low doses of L-Glu-L-Trp were more effective in inhibitingangiogenesis (groups 4-7) than higher doses (group 3). The inversedose-response profile, i.e., with lower activity at higher doses, isconsistent with previously observed performance of other biologicalresponse modifiers in this assay (e.g., IFN-α or IL-12).

All treatments were well tolerated and no weight loss or deaths wererecorded.

EXAMPLE 4 Anti-Tumor Activity of L-Glu-L-Trp: Sarcoma 180

Neovascularization is required for tumor growth. Anti-tumor activity ofL-Glu-L-Trp was evaluated at an independent contract researchorganization. Sarcoma 180 tumors (ATCC CCL-8 CCRF S-180 II) were inducedby injecting 2×10⁶ cells/0.1 ml im into each rear flank of Swiss-Webstermice. Groups consisted of 10 animals (20 tumors). L-Glu-L-Trp wasadministered in a single 0.1 ml dose of either 10 μg/kg, 75 μg/kg, 250μg/kg or 1000 μg/kg. Tumor size was evaluated by surgically removing andweighing the affected limbs, and comparing the weight with the weight ofnormal control (non-tumor) limbs. The first prophylactic drug regimen(PDR-1) consisted of 5 consecutive daily ip injections commencing on day−5 and ending of day −1. The second prophylactic drug regimen (PDR-2)consisted of 5 consecutive daily im injections to the left rear flank(tumor site) beginning on day −5 and ending on day −1. Sarcoma 180 cellswere injected im on day 0. Saline 0.1 ml served as the negative control.TABLE 4 Effect of L-Glu-L-Trp Treatments on Sarcoma 180 Tumor Size MeanPercent of Tumor Control Treatment Leg Weight (g) Weight^(a) Tumor GroupRegimen Dose (μg/kg) Left Right Left Right Weight^(b) 1A None 0 1.2 ±0.1 1.2 ± 0.2 0 0 — — (normal) 1B None 0 3.7 ± 0.6 3.5 ± 0.7 2.5 2.3 0 0(tumor) 2 PDR-1 10 4.2 ± 0.9 4.1 ± 0.7 3.0 2.9 120 126 3 PDR-1 75 4.2 ±0.8 4.2 ± 0.8 3.0 3.0 120 130 4 PDR-1 250 3.5 ± 1.0 3.1 ± 0.6 2.3 1.9 9283 5 PDR-1 1000 2.5 ± 0.7 2.2 ± 0.5 1.3 1.0 52 43 6 PDR-2 10 3.6 ± 0.73.8 ± 0.7 2.4 2.6 96 113 7 PDR-2 75 3.6 ± 0.7 3.5 ± 0.3 2.4 3.3 96 143 8PDR-2 250 3.0 ± 0.1 2.3 ± 0.5 1.8 1.1 72 48 9 PDR-2 1000 2.4 ± 0.5 2.5 ±0.5 1.2 1.3 48 57^(a)Mean Tumor weight = (mean leg weight treated - mean leg weightnormal control);^(b)Inhibition = (tumor weight treated/tumor weight control) × 100%

The results presented in TABLE 5 show that prophylactic treatments withL-Glu-L-Trp ip or im at doses of 250 μg/kg and 1000 μg/kg inhibitedsubsequent im tumor growth. Interestingly, it appeared possible toinvoke systemic inhibitor effects from treatments delivered at a localim site, because the im treatments delivered into the left flankinhibited subsequent tumor growth in the right flank (i.e., groups 8 and9). The results are consistent with the inhibition of neovascularizationobserved in Examples 2 and 3, above.

The present invention provides a substantially novel method forinhibiting neovascularization. While specific examples have beenprovided, the above description is illustrative and not restrictive.Many variations of the invention will become apparent to those of skillin the art upon review of this specification. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

1. A method of treating a subject having a pathologic conditioninvolving neovascularization comprising administering a pharmaceuticalpreparation comprising an R′-Glu-Trp-R″ dipeptide and a pharmaceuticallyacceptable carrier to the subject in an amount effective to inhibitneovascularization.
 2. The method of claim 1 wherein the R′-Glu-Trp-R″dipeptide is L-Glu-L-Trp.
 3. The method of claim 2 wherein the conditionis hemangioma.
 4. The method of claim 2 wherein the condition isvascularized malignant tumor or vascularized benign tumor.
 5. The methodof claim 2 wherein the condition is neovascularization in post-recoverycerebrovascular accident; neovascularization due to head trauma;restenosis following angioplasty; or neovascularization due to heat orcold trauma.
 6. The method of claim 2 wherein the condition isneovascularization associated with substance-induced neovascularizationof the liver, anglogenic dysfunction related to an excess of hormone;neovascular sequelae of diabetes; neovascular sequelae to hypertension;or chronic liver infection.
 7. The method of claim 2 comprisingadministering to the subject a dose of about 0.5 μg per 1 kilogram bodyweight to about 1 mg per 1 kg body weight.
 8. The method of claim 7wherein the effective amount is about 1 μg/kg to about 50 μg/kg bodyweight.
 9. The method of claim 7 wherein the dose is administered dailyover a period of 1 day to about 30 days.
 10. The method of claim 7wherein the pharmaceutical preparation is administered intramuscularlyor intranasally.
 11. The method of claim 2 comprising administering thepharmaceutical preparation in the form of an injectable solutioncontaining 0.001% to 0.01% of L-Glu-L-Trp.
 12. The method of claim 2comprising administering the preparation in a unit dose form comprisinga tablet, a suppository, a capsule, an eye film, an inhalant, a mucosalspray, a nose drop, an eye drop, a toothpaste, an ointment, or watersoluble based cream.
 13. The method of claim 12 wherein said unit doseform consists essentially of 0.01 mg of said R′-Glu-Trp-R″.
 14. Themethod of claim 2 further comprising administering to the subject avasoactive drug.
 15. The method of claim 14 wherein the vasoactive drugis an angiotensin converting enzyme (ACE) inhibitor or a potassiumchannel opener (PCO).
 16. The method of claim 2 wherein the subjectsuffers from a tumor wherein the method further comprises administeringa chemotherapeutic agent.
 17. The method of claim 2 wherein the subjectis not immune compromised.